RESUMEN
Intrinsic factors such as proteins modulate the fertilising ability of male gametes. We compared detergent-extracted sperm protein composition of bulls with different fertility indexes in order to highlight putative fertility markers of sperm. Frozen semen from 23 Holstein bulls with documented fertility was used. According to their 'fertility solution' (SOL), as calculated by the Canadian dairy network, bulls were divided into four groups: high fertility (HF) (SOL>3.0; n=6), medium-HF (2.9>SOL>2.0; n=5), medium-low fertility (-2.8>SOL>-4.9; n=8) and low fertility (LF; SOL<-5.0; n=4), with a SOL=0 being the average. Triton X-100 protein extracts from ejaculated spermatozoa were subjected to two-dimensional difference gel electrophoresis, and polypeptide maps were quantitatively analysed by ImageMaster software. Nine protein spots showed significant differences between the HF and LF groups, and eight of these proteins were identified by liquid chromatography-tandem mass spectrometry. T-complex protein 1 subunits epsilon and (CCT5 and CCT8), two isoforms of epididymal sperm-binding protein E12 (ELSPBP1), proteasome subunit alpha type-6 and binder of sperm 1 (BSP1) were more expressed in the LF group than in the HF group. On the other hand, adenylate kinase isoenzyme 1 (AK1) and phosphatidylethanolamine-binding protein 1 (PEBP1) were more expressed in the HF group than in the LF group. The presence and expression level of ELSPBP1, BSP1, AK1 and PEBP1 were confirmed by western blot. A linear regression model established that CCT5 and AK1 explained 64% (P<0.001) of the fertility scores. The reported functions of these proteins are in agreement with a putative involvement in defective sperm physiology, where lower or higher levels can jeopardise sperm ability to reach and fertilise the oocyte.
Asunto(s)
Bovinos , Detergentes/farmacología , Fertilidad/fisiología , Proteómica/métodos , Proteínas de Plasma Seminal/aislamiento & purificación , Proteínas de Plasma Seminal/metabolismo , Animales , Bovinos/fisiología , Cromatografía Liquida , Eficiencia , Electroforesis en Gel Bidimensional , Indicadores de Salud , Masculino , Proteínas de Plasma Seminal/análisis , Proteínas de Plasma Seminal/efectos de los fármacos , Espermatozoides/citología , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Disabled-1 (Dab1) is an intracellular adaptor protein that regulates migrations of various classes of neurons during mammalian brain development. Dab1 function depends on its tyrosine phosphorylation, which is stimulated by Reelin, an extracellular signaling molecule. Reelin increases the stoichiometry of Dab1 phosphorylation and downregulates Dab1 protein levels. Reelin binds to various cell surface receptors, including two members of the low-density lipoprotein receptor family that also bind to Dab1. Mutations in Dab1, its phosphorylation sites, Reelin, or the Reelin receptors cause a common phenotype. However, the molecular mechanism whereby Reelin regulates Dab1 tyrosine phosphorylation is poorly understood. RESULTS: We found that Reelin-induced Dab1 tyrosine phosphorylation in neuron cultures is inhibited by acute treatment with pharmacological inhibitors of Src family, but not Abl family, kinases. In addition, Reelin stimulates Src family kinases by a mechanism involving Dab1. We analyzed the Dab1 protein level and tyrosine phosphorylation stoichiometry by using brain samples and cultured neurons that were obtained from mouse embryos carrying mutations in Src family tyrosine kinases. We found that fyn is required for proper Dab1 levels and phosphorylation in vivo and in vitro. When fyn copy number is reduced, src, but not yes, becomes important, reflecting a partial redundancy between fyn and src. CONCLUSIONS: Reelin activates Fyn to phosphorylate and downregulate Dab1 during brain development. The results were unexpected because Fyn deficiency does not cause the same developmental phenotype as Dab1 or Reelin deficiency. This suggests additional complexity in the Reelin signaling pathway.
Asunto(s)
Encéfalo/crecimiento & desarrollo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Animales , Encéfalo/embriología , Encéfalo/metabolismo , Moléculas de Adhesión Celular Neuronal/efectos de los fármacos , Moléculas de Adhesión Celular Neuronal/fisiología , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Proteínas de la Matriz Extracelular/efectos de los fármacos , Proteínas de la Matriz Extracelular/fisiología , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Ratones Mutantes , Proteínas del Tejido Nervioso/genética , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Tirosina Quinasas/efectos de los fármacos , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-abl/genética , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteínas Proto-Oncogénicas c-fyn , Proteínas Proto-Oncogénicas c-yes , Proteína Reelina , Proteínas de Plasma Seminal/efectos de los fármacos , Proteínas de Plasma Seminal/genética , Proteínas de Plasma Seminal/metabolismo , Serina Endopeptidasas , Transducción de Señal , Tirosina/metabolismo , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Human sperm capacitation involves complex signal transduction mechanisms during which double phosphorylation of the threonine-glutamine-tyrosine motif (P-Thr-Glu-Tyr-P) occurs in some sperm proteins. The objective of this study was to investigate the regulation of this process. Fetal cord serum ultrafiltrate (FCSu), follicular fluid ultrafiltrate (FFu), progesterone and a combination of N(6),2'-O-dibutyryl cAMP (dbcAMP; cell permeant analogue of cAMP) and 3-isobutyl-1-methylxanthine (IBMX; phosphodiesterase inhibitor) were used as inducers of capacitation alone or in combination with inhibitors of protein kinase A (H89), protein kinase C (chelerythrine), protein tyrosine kinase (tyrphostin A47, PP2) and of dual specificity kinase (MEK-like kinases; PD98059). The level of P-Thr-Glu-Tyr-P in sperm proteins of 80 and 105 kDa during capacitation induced by FCSu, FFu and progesterone was regulated by a similar signal transduction pathway and involved receptor type protein tyrosine kinase and dual specificity kinase (MEK or MEK-like) but not protein kinase A or C. However, the level of P-Thr-Glu-Tyr-P in these sperm proteins during capacitation induced by dbcAMP+IBMX was mainly mediated through protein kinase A and C and receptor type protein tyrosine kinase, but not by dual specificity kinase. In conclusion, human sperm capacitation induced by some biological and pharmacological agents is regulated through very different signal transduction pathways.